How enzymes function as catalysts and how they are regulated at the molecular and hormonal level is of fundamental importance to the biologist. The studies proposed in this application involve the investigation of enzymes involved in glycolysis and gluconeogenesis and their regulation. In the case of glycolysis, experiments will be centered around two isozymes, brain and muscle hexokinase. The activity of the latter phosphotransferase has been shown to decrease in streptozotocin-induced diabetes and returns to normal upon the administration of insulin. We have shown that the rate of degradation of muscle hexokinase in the diabetic rat is three times greater than in the normal animal. We propose to investigate the rates of synthesis and degradation in insulin treated diabetic animals and the rate of synthesis of muscle hexokinase in normal and diabetic animals. Brain hexokinase is the pacemaker of glycolysis in the brain and red blood cells, and its activity is controlled by its products and inorganic phosphate. We plan to investigate the mechanism of brain hexokinase regulation using kinetic and nuclear magnetic resonance techniques. The gluconeogenic enzyme we propose to investigate is fructose 1,6-bisphosphatase. It has recently been demonstrated that fructose 2,6-bisphosphate is a potent inhibitor of the phosphatase and an activator of the glycolytic enzyme, phosphofructokinase. We have preliminary evidence showing how the inhibitor affects the activity of fructose 1,6-bisphosphatase at the molecular level and we plan to pursue these investigations using nuclear magnetic resonance procedures. In addition, we expect to investigate the kinetic mechanism of action of the enzyme responsible for the biosynthesis of the inhibitor, fructose-6-P 2 kinase, and finally, we propose to study its regulation using kinetic protocols. The same procedures will be used to investigate the mechanism and regulation of fructose 1,6-bisphosphatase.